Organelle-Level Labile Zn²⁺ Mapping Based on Targetable Fluorescent Sensors

Abstract

Although many Zn 2+ fluorescent probes have been developed, there remains a lack of consensus on the labile Zn 2+ concentrations ([Zn 2+ ]) in several cellular compartments, as the fluorescence properties and zinc affinity of the fluorescent probes are greatly affected by the pH and redox environments specific to organelles. In this study, we developed two turn-on-type Zn 2+ fluorescent probes, namely, ZnDA-2H and ZnDA-3H, with low pH sensitivity and suitable affinity (K d = 5.0 and 0.16 nM) for detecting physiological labile Zn 2+ in various cellular compartments, such as the cytosol, nucleus, ER, and mitochondria. Due to their sufficient membrane permeability, both probes were precisely localized to the target organelles in HeLa cells using HaloTag labeling technology. Using an in situ standard quantification method, we identified the [Zn 2+ ] in the tested organelles, resulting in the subcellular [Zn 2+ ] distribution as [

Mobile zinc as a modulator of sensory perception

“…Examples include a benzylguanine moiety for alkylation of human O 6 ‐alkylguanine transferase (SNAP‐tag) and a chloroalkane linker for reaction with a haloalkane dehalogenase (HaloTag ® ) [ 46 , 47 ]. These methods can also be used in combination with other zinc sensors for multiwavelength imaging [ 48 ].…”

“…In cells and live tissue slices, TPA removes zinc from fluorescence sensors more than twice as fast as TPEN [ 93 ]. The lower cellular toxicity associated with TPA makes it attractive for studies in a wide variety of biological contexts [ 48 , 94 , 95 , 96 , 97 , 98 ]. Although there is some evidence that TPA chelates metal ions other than zinc(II), particularly copper(I), it does not appear to substantially affect the concentrations of these ions at the less toxic and membrane‐permeable levels where it has typically been applied [ 99 , 100 ].…”

An Endoplasmic Reticulum-Targeted Ratiometric Fluorescent Molecule Reveals Zn2+ Micro-Dynamics During Drug-Induced Organelle Ionic Disorder

“…Fluorescent probe method has been widely used to detect ionic dynamics in living cells due to its advantages of low toxicity, easy preparation, and in situ non-destructive detection ( Chen et al, 2015 ; Li et al, 2020 ; Chen et al, 2021 ; Fang et al, 2021a ; Liu et al, 2021 ; Wang and Diao, 2022 ; Wang et al, 2022 ). Therefore, the use of fluorescent probes to study Zn 2+ in ER provides a powerful tool for the detection and treatment of ER stress-related diseases by regulating the Zn 2+ level ( Chabosseau et al, 2018 ; Huang et al, 2021 ; Liu et al, 2022 ). Although many Zn 2+ fluorescent probes have been developed, most of them show turn-on response and are difficult to use for in vivo detection ( Fang et al, 2021b ; Qi et al, 2021 ; Wang et al, 2021 ).…”

Fluorogenic and genetic targeting of a red-emitting molecular calcium indicator

“…[6][7][8] The most commonly used SLP tags, SNAPtag and HaloTag, have for instance been used to control the subcellular localization of Ca 2+ or Zn 2+ probes. [9][10][11][12] However, even with an efficient targeting reaction, one has to get rid of the unbound probes that may yield an unspecific signal. Metal cations sensors often involve cell-impermeant carboxylate chelating groups masked as ester functions and the excess of dye cannot always be easily washed away after esterase hydrolysis in cells.…”